Bacillus strain for breaking down moenomycins

ABSTRACT

A new Bacillus species and the appropriate enzymes obtained therefrom can be used to break down phosphoglycolipid antibiotics. The breakdown products of moenomycins display antibiotic activity or can be used as building blocks for the synthetic preparation of transglycosylase inhibitors.

This application is a continuation of application Ser. No. 08/202,760,filed Feb. 28, 1994, now abandoned and division of application Ser. No.08/048,511, filed Apr. 20, 1993, now U.S. Pat. No. 5,315,038; which is acontinuation-in-part application of Ser. No. 07/927,886, filed Aug. 11,1992, now U.S. Pat. No. 5,206,405; which is a continuation applicationof Ser. No. 07/617,635, filed Nov. 26, 1990, abandoned; which is adivisional application of Ser. No. 07/395,790, filed Aug. 18, 1989,abandoned; and a continuation-in-part application of Ser. No.07/938,599, filed Sep. 3, 1992, now U.S. Pat. No. 5,260,206; which is acontinuation-in-part application of Ser. No. 07/762,262, filed Sep. 20,1991, abandoned; which is a continuation application of Ser. No.07/711,708, filed Jun. 7, 1991, abandoned; which is a continuationapplication of Ser. No. 07/395,790, filed Aug. 18, 1989, abandoned.

Moenomycin A is the main component of Flavomycin® which is used inlivestock nutrition. Like other known phosphoglycolipid antibiotics itinhibits the biosynthesis of the peptidoglycan framework of thebacterial cell wall. Closer investigations showed that thetransglycosylation reaction of the penicillin-binding protein 1b of E.coli is inhibited by these substances (G. Huber, in F. E. Hahn (ed.)Antibiotics, Vol. 1, 135-153, Berlin: Springer, 1979). Attempts atspecific enzymatic or microbial breakdown of phosphoglycolipidantibiotics have hitherto failed.

Several attempts to chemically synthesize biologically active structuralanalogues of the moenomycin-type antibiotics have been unsuccessful.

H. Hohgardt et al., Tetrahedron, 44(18):5771-5790 (1988), were able toprepare disaccharides connected via phosphorous acid and glycerol acidto the saturated moenocinol. These compounds were devoid of anyantibiotic activity. They differ from an active disaccharide moenomycinanalogue (prepared from moenomycin by chemical degradation as describedin P. Welzel et al., Tetrahedron, 43:585-598 (1987)), in (1) having agalacturonic acid configuration instead of a moenuronic acidconfiguration, (2) lacking double bonds in the lipid (alkyl) moiety, and(3) bearing a carboxylic acid at position 6 of moenuronic acid ratherthan a carboxamide.

U. Moller et al., Tetrahedron, 49:1635-1648 (1993)), introduced themissing carboxamide into the substructure at position 6 of moenuronicacid, resulting in a molecule which differs from an active analogue onlyin (1) having a galacturonic acid configuration instead of a moenuronicacid configuration, and (2) lacking double bonds in the alkyl chain.Surprisingly, this compound was also antibiotically inactive.

Surprisingly, a new Bacillus species which is able to cleave thephosphoglycolipid antibiotics to defined end products has now beenisolated from a contaminated fermenter for the preparation of flavomycinusing Streptomyces ghanaensis. These end products have antibioticactivity or can be used as building blocks in the synthesis of newtransglycosylase inhibitors.

Hence, the invention relates to:

1. Bacillus spec. DSM 4675 and the variants and mutants thereof.

2. The cleavage product of moenomycin A with the formula I. ##STR1## 3.The cleavage product of the phosphoglycolipid antibiotics with thegeneral formula ##STR2## in which R¹ is hydrogen or a phosphono group[-PO(OH)₂ ] and R² is a (C₅ to C₅₅)-alkyl group which can be branched orunbranched, saturated or unsaturated.

4. The enzymes with whose aid the phosphoglycolipid antibiotics can becleaved at the phosphoglycosidic linkage, or the cleavage productsspecified under 3. can be cleaved at the monophosphate ester linkage.

5. A process for the preparation of the breakdown products specifiedunder 2. and 3., which comprises incubating phosphoglycolipidantibiotics with Bacillus spec. DSM 4675.

6. The use of the substances specified under 2. and 3. as building blockfor the synthetic preparation of transglycosylase inhibitors or assubstance having antibiotic activity.

The invention will be described in detail hereinafter, especially in thepreferred embodiments. It is furthermore defined in the claims. Bacillusspec. was deposited with the number DSM 4675 under the provisions of theBudapest Treaty at the Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (German Microorganism and Cell Culture Collection) inBraunschweig, PRG, on Jun. 23, 1988. The characteristics of the strainmay be said to be the following:

1. Taxonomic properties of Bacillus sp. DSM 4675

A) Morphology

motile rods; up to 5 μm long; some in short chains

terminal spore; sporangium swollen

Gram-positive.

B) Growth on various media (28° C.; 48 hours)

1. Antibiotic medium 3 (Difco)

rough, lobed colonies of diameter 1-2 mm

2. Luria broth (Bacto tryptons 10 g/l; Bacto yeast 5 g/l; NaCl 5 g/l

smooth, glossy round colonies of diameter 2-3 mm; opaque

3. Nutrient broth (Difco) white, glossy colonies with irregular margin

4. Christensen urea agar (Difco) growth positive

5. McConkey agar (Difco) growth positive

6. BROLAC agar (lactose) (Difco) growth positive

7. Simmons citrate agar (Difco) growth negative

8. No growth in the presence of 7 or 10% NaCl in a peptone/meat extractmedium (Difco)

C) Physiological properties

    ______________________________________                                         1.     Oxidase           +                                                    2.     Catalase          +                                                    3.     Hemolysis         -                                                    4.     Aminopeptidase    -                                                    5.     Nitrate reduction -                                                    6.     Phenylalanine deaminase                                                                         -                                                    7.     Growth at 30° C.                                                                         +                                                           40° C.     +                                                           50° C.     +                                                    8.     Anaerobic growth                                                              solid             -                                                           liquid            -                                                    9.     Gas formation from glucose                                                                      -                                                   10.     Indole formation  -                                                   11.     Arginine dihydrase                                                                              -                                                   12.     Urea breakdown    -                                                   13.     Esculin hydrolysis                                                                              +                                                   14.     Gelatin breakdown -                                                   15.     β-Galactosidase                                                                            +                                                   16.     Lysine decarboxylase                                                                            -                                                   17.     Ornithine decarboxylase                                                                         -                                                   18.     H.sub.2 S production                                                                            -                                                   19.     Tryptophan deaminase                                                                            -                                                   20.     Alkal. phosphatase                                                                              -                                                   21.     Voges-Proskauer reaction                                                                        -                                                   ______________________________________                                    

D) Fermentation of carbohydrates

    ______________________________________                                        C source        Assimilation                                                                             Acid formation                                     ______________________________________                                        Adipate         -                                                             Adonitol        -                                                             Arabinose       +          +                                                  Caprate         -                                                             Citrate         -                                                             Dulcitol        -                                                             Fructose        +          +                                                  Galactose       -                                                             Gluconate       +                                                             Glucose         +          +                                                  Inositol        -                                                             Lactose         +          +                                                  Malate          -                                                             Malonate        -                                                             Maltose         +          +                                                  Mannitol        +          +                                                  Mannose         +          +                                                  Melibiose       +          +                                                  Phenylacetate   -                                                             Raffinose       +          +                                                  Rhamnose        -                                                             Sucrose         +          +                                                  Salicin         -                                                             Sorbitol        -                                                             Trehalose       +          +                                                  Xylitol         -          -                                                  Xylose          +          +                                                  N-Acetyl-glucosamine                                                                          -          +                                                  ______________________________________                                    

Taking account of taxonomic features and with the aid of "Bergey'sManual of Systematic Bacteriology" (Vol. 2, Williams and Wilkins publ.,Baltimore, 1986) the strain can be assigned to the genus Bacillus. Todetermine the species, parallel comparative examinations of typecultures of Bacillus macerans, circulans, lentus, alcalophilus,stearothermophilus, licheniformis, polymyxa and fastidiosus were carriedout. All the comparison strains showed distinct differences fromBacillus spec. DSM 4675. Nor were any of these strains able to breakdown phosphoglycolipid antibiotics, especially moenomycin A. Theconclusion to be drawn from this is that the strain DSM 4675 is a newspecies.

The invention also relates in each case to the mutants and variantswhich, as is known, may arise spontaneously or be generated by treatmentwith physical agents, for example irradiation, such as ultraviolet orX-rays, or with chemical mutagens such as, for example, ethylmethanesulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or2-hydroxy-4-methoxy-benzophenone (MOB).

Suitable and preferred as carbon source for the aerobic fermentation ofBacillus spec. DSM 4675 are assimilable carbohydrates and sugaralcohols, such as glucose, lactose or mannitol, as well ascarbohydrate-containing natural products such as malt extract. Suitableand preferred nitrogen-containing nutrients are: amino acids, peptidesand proteins, as well as the breakdown products thereof, such as peptoneor tryptone, furthermore meat extracts, milled seeds, for example ofcorn, beans, soybean or the cotton plant, disillation residues from theproduction of alcohol, meat meals or yeast extracts, as well as ammoniumsalts and nitrates. The nutrient solution may additionally contain, forexample, chlorides, carbonates, sulfates or phosphates of the alkalimetals or alkaline earth metals, iron, zinc and manganese as additionalinorganic salts.

The growth of the microorganism and the formation of the enzymesnecessary for the breakdown reactions according to the invention isparticularly good in a nutrient medium containing corn starch, soybeanmeal, sucrose, glycerol, peptone and/or corn steep as carbon andnitrogen sources.

The fermentation is carried out aerobically, that is to say, forexample, submerged with shaking or stirring in shaken flasks orfermenters, where appropriate introducing air or oxygen. Thefermentation can take place in a temperature range from approximatelyroom temperature to 50° C., preferably at about 35° to 37° C. Theculturing time is generally 24 to 48 hours.

The cultures of Bacillus DSM 4675 obtained in this way, or preparationsthereof, can be used to cleave the phosphoglycolipid antibiotics. Theseinclude, in particular, the antibiotics of the moenomycin group, forexample pholipomycin¹), the prasinomycins²), the diumycins(macarbomycins)³) esanchomycin, prenomycin and teichomycin, and otherstructurally related substances which have a correspondinglyfunctionalized phosphoglyceric acid [1) S. Takahashi et al., TetrahedronLett. 1983, 499 2) F. L. Weisenborn et al., Nature 213, 1092 (1967) 3)S. Takahashi et al., J. Antibiot. 26, 542 (1973)].

The enzymes are particularly preferably used to break down themoenomycins, for example Flavomycin.

When Bacillus cells are used it is advantageous to permeabilize theletter, for example with cetyltrimethylammonium salts. It is likewisepossible to use protein isolates from the Bacillus cells, or enzymeextracts which have been partially enriched, for example, by salting-outor chromatography or, of course, the purified enzyme. It is furthermorepossible to employ the enzyme and cells in free or immobilized form.

The enzymatic breakdown is depicted in the following diagram usingmoenomycin A as an example. ##STR3##

It is evident from this diagram that two enzymes are needed to preparethe cleavage products. An enzyme, which the inventors have calledmoenomycinase, is required to cleave the phosphoglycosidic linkage.Moenomycinase is associated with the cytoplasmic membrane of Bacillusspec. DSM 4675 and can be obtained from the microorganism by enzymeisolation methods known per se.

For example, moenomycinase bound to membranes of Bacillus spec. DSM 4675can be solubilized with the detergent Triton X-100 (1-3%) as describedin Example 2A. Moenomycinase can also be isolated without the use ofdetergent by extensive ultrasound sonication. For example, cellssuspended in buffer (20 mM potassium phosphate, pH 8.0, with 0.1 mMCoCl₂) were sonicated for 1-2 minutes. After centrifugation at 20min×20,000 g, 50-60% of the enzyme activity was found in thesupernatant. Alternatively, some activity can be solubilized withcetyltrimethylammonium salts. For example, when cells were incubated (1g/2 ml) in 0.1% cetyltrimethylammonium salts (0.1%), 10% of the enzymeactivity was found in the supernatant after centrifugation for 20min×20,000 g.

The molecular weight of moenomycinase is 230,000±10,000 Dalton.Moenomycinase is activated by Co⁺⁺, Ni⁺⁺, Mn⁺⁺, Ca⁺⁺ and Mg⁺⁺, and canbe inhibited by formalin, EDTA, Cephalosporin C, and7-aminocephalosporin acid (7-ACA).

Moenomycinase has a pH optimum of about 8.0-8.5, in particular 8.2-8.3.The temperature optimum of the enzyme is 45°-55° C., in particular49°-51° C. Moenomycinase has a K_(m) value for moenomycin A of 4-10millimolar.

Two cleavage products are obtained. Cleavage product I comprises thesugar component of the phosphoglycolipid antibiotics. Also obtained isthe cleavage product with the general formula II ##STR4## in which R¹ isa phosphono group and R² is a (C₅ to C₅₅)-alkyl group, preferably a (C₁₀to C₃₀)-alkyl group, in particular a (C₂₀ to C₂₅)-alkyl, each of whichcan be branched or unbranched, saturated or unsaturated, preferablybranched and unsaturated.

Moenomycins are preferably employed as substrates, so that the resultingcleavage products are the substances corresponding to the compound MC,as well as the compounds MB and MA (see formula diagram).

Moenomycinase is activated by metal ions such as Co⁺⁺, Ni⁺⁺, Mn⁺⁺ andMg⁺⁺ (0.05-1.0 mM). Although greatest activation is seen with Ni⁺⁺, thecurrently preferred ion for activation of moenomycinase for commercialdevelopment is Co⁺⁺ (0.10 mM).

Another enzyme is required for the dephosphorylation of thephosphoglyceric acid lipid, which is obtained by incubation withmoenomycinase, of the general formula II in which R¹ is hydrogen, andcan also be obtained from the microorganism according to the invention.The inventors have called this enzyme MBase. MBase can likewise beisolated from the microorganism by methods known per se. For example,the cells are disrupted with ultrasound, and the resulting crude extractis further enriched either by ammonium sulfate fractionation (25-55%saturation) or ultracentrifugation. This is followed by dialysis. Themoenomycinase and MBase are finally separated by chromatography.

The MBase is increasingly inactivated at temperatures above 37° C. aswell as when the pH falls in the acid pH range below pH 5.

The cleavage of the moenomycinase, as well as of the phosphoglycolipidantibiotics can, as already mentioned, be carried out with whole cellsor enzyme isolates.

The reaction is generally carried out in aqueous medium at a pH of about5.5-8.5, preferably pH 7-8. The reaction time is generally 5-48 hours,preferably about 24 hours. The reaction temperature can be from 4° to60° C., preferably 30° to 37° C. The substrate concentration ought to bein the range from 0.1 to 5%, preferably 1 to 2%.

It is still possible to carry out the reaction at temperatures or pHvalues which are higher or lower than stated. However, the moenomycinaseis then less active.

The reaction products resulting from moenomycin A are the substances MBand MC depicted in the diagram. The product MA can be obtained byincubation of MB with MBase at 30° to 37° C., preferably at 35° to 37°C., and pH 5.5 to 8.5, preferably pH 6 to 8, over a period of about 24hours.

A process for the preparation of MA is described in European PatentApplication EP 503419. The said reaction products can be used asantibiotic (for example MB) or as building blocks for the synthesis oftransglycosylase inhibitors (for example MA and MC).

The invention is described in more detail by means of examples. Unlessstated otherwise, percentage data relate to weight.

EXAMPLE 1 Maintenance of the Bacillus Spec. DSM 4675 Strain

Bacillus spec. DSM 4675 is maintained on the following solid nutrientmedium (medium 1):

    ______________________________________                                        Bacto tryptone (Difco)                                                                           10 g/l                                                     Yeast extract (Difco)                                                                             5 g/l                                                     NaCl                5 g/l                                                     Agar               15 g/l                                                     pH 7.2                                                                        ______________________________________                                    

The medium is distributed over test tubes and sterilized at 121° C. for30 minutes, then cooled, inoculated with the culture and incubated at37° C. for 2-3 days.

The grown culture is rinsed off to provide the inoculum for thefollowing, moenomycin-containing main culture (medium 2):

    ______________________________________                                        Corn starch            40    g/l                                              Soybean meal           35    g/l                                              Sucrose                10    g/l                                              CaCO.sub.3             8     g/l                                              Corn steep             4     g/l                                              CoSO.sub.4             20    mg/l                                             ® Genapol (alkyl polyglycol ester                                                                5     ml/l                                             Moenomycin A           3     g/l (sterile                                                                  filtered)                                        pH 7.6                                                                        ______________________________________                                    

300 ml Erlenmeyer flasks each containing 30 ml of this medium areinoculated and then incubated at 37° C. and 190 rpm for 8-48 hours.Analysis of the culture filtrate by thin-layer chromatography shows thatthe compounds MA, MB and MC are detectable as cleavage products ofmoenomycin, and that towards the end of the reaction there has beencomplete disappearance of the moenomycin employed.

EXAMPLE 2 Preparation of Cell-free Extracts

To prepare cell-free extracts, Bacillus spec. DSM 4675 is cultured in afermenter. For this, cells are rinsed off the agar plate to provide a 10ml inoculum for a preculture (500 ml of medium 2 without Flavomycin in a2 l Erlenmeyer flask) which is then incubated at 37° C. and 190 rpm for24 hours.

A 12 l laboratory fermenter containing 9 l of medium 3 is used for themain culture stage:

    ______________________________________                                        Peptone                 12.5   g/l                                            Glycerol                20.0   tg/l                                           Citrate                 2.0    g/l                                            K.sub.2 HPO.sub.4       1.5    g/l                                            MgSO.sub.4 × 7H.sub.2 O                                                                         0.5    g/l                                            FeCl.sub.3 × 6H.sub.2 O                                                                         0.04   g/l                                            Desmophen (propylene glycol)                                                                          5.0    ml/l                                           pH 6.8                                                                        ______________________________________                                    

This is inoculated with 500 ml of preculture and incubated at 37° C.,300 rpm and an aeration rate of 0.5 vvm for 24 hours.

The grown culture is centrifuged, and the cell paste is resuspended inpotassium phosphate buffer (pH 7.0) 50 mM (1 g of wet cells+2 ml ofbuffer). The cells are then disrupted with ultrasound, a French Press®or Dyno Mill®, and the resulting crude extract is used for theconversion.

In a test mixture containing 100 μl of crude extract, 12 mg ofmoenymycin and 900 μl of potassium phosphate buffer (pH 8.0) 50 mM thereis within 7-24 hours at 37° C. 50% breakdown of the substrate employed.The reaction products found are MA, MB and MC.

EXAMPLE 2A Isolation of Moenomycinase

Moenomycinase has been isolated from Bacillus spec. DSM 4675 as follows:

Cells grown as in Example 2 were suspended at a concentration of 1 gcells/2 ml in 20 mM potassium phosphate buffer, pH 8.0, containing 0.1mM CoCl₂. The suspended cells were sonicated for 20 seconds. Aftersonication, the suspension was centrifuged for 20 min×20,000 g. Thesupernatant was discarded and the pellet resuspended in the same bufferto which has been added 1% Triton X-100 and incubated for 1 hr at roomtemperature with stirring. The suspension was then centrifuged for 2.5hrs×100,000 g. After such centrifugation, 90-95% of the moenomycinaseactivity was found in the supernatant.

Moenomycinase solubilized using Triton X-100, as described above, wasfurther purified by (NH₄)₂ SO₄ precipitation. As (NH₄)₂ SO₄concentration was increased from 0% to 40%, the moenomycinaseprecipitated at 30-40% and entered a fatty phase that floats above theaqueous phase. The aqueous phase was discarded, and the fatty phase wasdissolved in 20 mM Tris HCl buffer, pH 8.0, containing 0.1 mM CoCl₂ and0.1% Triton X-100.

The dissolved enzyme was further purified by chromatography. For anionexchange chromatography, the enzyme was bound to a column of DEAE-52(Whatman) or MonoQHR-5-5 (Pharmacia) and then eluted with 0.2-1.0M NaCl.Presence of moenomycinase in the fractions was monitored by measuringthe conversion of moenomycin A to MC, as described in Example 3, below.Further purification was achieved using hydrophobic chromatography. Theenzyme was bound to phenylsepharose (Pharmacia), and was then elutedwith 40% methanol. The enzyme was concentrated by ultrafiltration usingan ultrafiltration membrane (Millipore) which retains molecules withmolecular weights≧10,000 Dalton.

The moenomycinase was further purified by molecular sieve chromatographyusing a Sephacryl S-200HR (Pharmacia). The enzyme was separated in theSephacryl chromatography column in 20 mM Tris HCl or potassium phosphatebuffer, pH 8.0, containing 0.1 mM CoCl₂ and 0.1% Triton X-100 and washedwith additional buffer. By comparison with the elution profiles of aseries of standard proteins of differing molecular weight, the molecularweight of the moenomycinase was determined to be 230,000±10,000 Dalton.If desired, the purity of the isolated moenomycinase fraction can bechecked using SDS gel chromatography.

EXAMPLE 3 Preparative Conversion of Moenomycin A

Preparative conversions are carried out in a 12 l fermenter containing8.2 l of potassium phosphate buffer (pH 8.0) 50 mM, 800 ml of crudeenzyme extract and 100 g of moenomycin A at 37° C. and 100 rpm. Theprogress of the conversion is followed by TLC. The entire reactionmixture is freeze-dried after 8-48 hours. The moenomycin breakdown isgenerally 40-60% (UV analysis from the TLC). The resulting reactionproducts are MA, MB and MC.

EXAMPLE 4 Preparation of the Breakdown Products

100 g of freeze-dried mixture from the enzymatic conversion were takenup in 2 l of water and extracted twice with 2 l of ethyl acetate eachtime. Centrifugation was necessary for complete phase separation in thiscase. The combined organic extracts were dried over sodium sulfate,filtered and evaporated to dryness. 0.4 g (0.4%) of a dark brown oil wasobtained, and thin-layer chromatography in the system n-butanol/aceticacid/water=3/1/2 on silica gel (Merch 60 F254 aluminum TLC sheets),spraying with molybdatophosphoric acid/cerium(IV) sulfate color reagent(abbreviated to PMS reagent hereinafter), showed that it comprisedmainly component MA (Rf value=0.85).

The remaining aqueous phase was then extracted twice with 2 l ofn-butanol each time. Once again, centrifugation was necessary for phaseseparation. The combined butanol phases were then concentrated as far aspossible, and the residue was taken up in a little water and finallyfreeze-dried. 7.4 g (7.4%) of a yellow powder were obtained and werefound on examination by thin-layer chromatography (using theabovementioned conditions and the same detection) to comprise mainly thecomponent MB (Rf=0.52).

The aqueous phase from which non-polar substances had been removed inthis way was freeze-dried. The amount of residue resulting from this was76.9 g (76.9% with a total amount of 85%). Thin-layer chromatographyshowed that this pale yellow powder was composed of a polar mainsubstance, called MC (Rf=0.1), remaining moenomycin A and by-products.

The crude products of components MA, MB and MC obtained in this way werefurther purified as follows.

EXAMPLE 5 a) Purification of the Breakdown Product MA

400 mg of MA crude product were chromatographed on 120 g of silica gel(Merck 60, 15-40 mcm) which had been adjusted to a pH of 7.5. (Thecolumn material had been pretreated in the following way for thispurpose: the silica gel was stirred in 500 ml of 2N HCl for one hour,then filtered off with suction and washed to neutrality. The pH was thenadjusted to 7.5 with 1N NaOH, and finally washing with 2 l of water and500 ml of methanol was carried out. The material pretreated in this waywas dried and activated at 120° C. overnight.) Chloroform/ethanol=1/1was used as eluent. The substance was loaded onto the column in 3 ml ofsolvent mixture, and 216 fractions each of 2.5 ml were collected. UsingTLC analysis (TLC plates and detection as in Example 1, solvent systemas for column eluent), fractions 115-175 were combined, dried on sodiumsulfate and finally evaporated to dryness. 27 mg of spectroscopicallypure MA were obtained.

b) Purification of the Breakdown Product MB

3.1 g of MB-containing crude product from the extraction werechromatographed on 500 g of silica gel which had been adjusted to a pHof 7.5 using the process explained in Example 2. Using a medium-pressurechromatography system (MPLC), chloroform/ethanol/water=4/7/1.5 was usedfor the elution at a flow rate of 10 ml/min and a pressure of 2-5 bar.After a fore-run of 600 ml, 220 fractions each of 10 ml were collected,combining on the basis of the TLC. Besides mixed fractions containingMB, fractions 90-170 yielded 1.28 g of pure MB after evaporation, takingup in water and freeze-drying.

c) Purification of the Breakdown Product MC

2.2 g of polar crude product from the aqueous phase of the extractionwere likewise chromatographed under pressure (MPLC). 500 g of silica gelwith a pH of 7.5 were employed (process in Example 2), and the eluentused was ethyl acetate/i-propanol/water=4/5/5. The amount to be loadedwas suspended in methanol with 15 g of silica gel, the solvent wasevaporated off, and the support treated in this way was introduced intoa precolumn, and then elution was carried out at a flow rate of 5 ml/minunder a pressure of 2-4 bar. A fore-run of 940 ml was followed byfractionation in 250 fractions each of 10 ml. The fractions were testedby thin-layer chromatography in the system ethylacetate/i-propanol/water=1/1/1 using PMS color reagent and detection ofthe UV absorption at 254 nm, and were combined. Besides mixed fractions,concentration of fractions 120-190 to the aqueous phase and subsequentfreeze-drying revealed 1.3 g of pure MC, which was investigated byspectroscopy.

EXAMPLE 6

Elucidation of the structures of the products MA and MB obtained frommoenomycin A by enzymatic breakdown.

(The numbers of the structures relate to the formula diagram on page 19)

The structure deduced for MA was 1a. The assignment of the structure isbased on the ¹³ C NMR spectrum of 1a. Reaction of 1a with diazomethaneyielded the methyl ester 1b which is characterized by a ¹ H NMR and anEI mass spectrum. The structure deduced for MB on the basis of ¹³ C andFAB mass spectrum was 2. Hydrogenation of 2 yielded the decahydroderivative 3a which reacted with diazomethane to give 3b, which hadalready been obtained previously from moenomycin A by another route. Itis consistent with the proposed structures that it was possible toconvert 2 (MB) enzymatically into 1a (MA).

Description of the Experiments

1a

¹³ C NMR (100.6 MHz, CD₃ OD): moenocinol moiety: δ=67.5 (C-1), 123.5(C-2), 141.6 and 141.8 (C-3 and C-7), 32.3 and 32.5 (C-4 and C-5), 126.7(C-6), 35.9 (C-8), 40.9 (C-9), 30.7 (C-10), 151.1 (C-11), 33.4 (C-12),122.7 (C-13), 137.3 (C-14), 36.4 (C-15), 27.7 (C-16), 125.3 (C-17),132.2 (C-18), 25.9 (C-19), 17.8 (C-20), 16.1 (C-21), 109.2 (C-22), 27.3(C-23 and C-24), 23.8 (C-25). Glyceric acid moiety: δ=175.9 (C-1), 80.6(C-2), 64.1 (C-3). C₂₈ H₄₆ O₄ (446.6 )

1b

1a was converted into the H⁺ form in aqueous solution using ®Dowex 50(H⁺ form). 1a (H⁺ form, 18.5 mg, 0.04 mmol) was dissolved in methanol (3ml) and, at 0° C., excess ethereal diazomethane solution was added. Thereaction mixture was maintained at 0° C. for 2 h and at 20° C. for 12 hand then evaporated to dryness. Column chromatography (5 g of SiO₂,petroleum ether/ethyl acetate 2:1) yielded 1b (3 mg).

¹ H NMR (80 MHz, CDCl₃): δ=0.96 (s, 6H, CH₃ -23 and CH₃ -24), 1.61 (s,6H), 1.68 (s, 3H), 1.73 (s, 3H) (4×CH₃), 1.90-2.12 (allyl Hs), 2.62(broad d, J=7 Hz, CH₂ -12), 3.78 (s, OCH₃), 3.60-4.30 (OCH₂ and OCHsignals), 4.62 (broad s, CH₂ -22), 4.87-5.47 (olefinic Hs). -C₂₉ H₄₈ O₄(460.7), MS: m/z (%)=460 (0.03), 271 (10), 230 (19), 199 (68), 43 (100).

¹³ C NMR (100.6 MHz, D₂ O): moenocinol moiety: δ=68.6 (C-1), 124.5(C-2), 142.9 (C-3), 34.6 (C-4), 34.0 (C-5, C-10), 128.1 (C-6), 143.6(C-7), 37.8 (C-8), 44.1 (C-9), 151.4 (C-11), 37.2 (C-12), 123.5 (C-13),138.3 (C-14), 42.2 (C-15), 29.1 (C-16), 126.9 (C-17), 133.1 (C-18), 28.0(C-19), 19.9 (C-20), 18.2 (C-21), 111.2 (C-22), 29.7 (C-23, 24), 25.9(C-25). Glyceric acid moiety: δ=179.0 (C-1), 81.8 (C-2), 68.6 (C-3).-C₂₈ H₄₇ O₇ P (526.7), FAB-MS (matrix: DMSO/glycerol): m/z=615 (M-3H+4Na)⁺, 593 (M-2H+3Na)⁺, 571 (M-H+2Na)⁺, 492, 267, 231, 185, 165, 143,115.

3a

2 (14.9 mg, 28.3 μmol) and PtO₂ (4 mg) were stirred in methanol (3 ml)and acetic acid (50 μl) in an H₂ atmosphere under normal conditions for3 days. The catalyst was filtered off, and evaporation yielded 3a (13.5mg). -C₂₈ H₅₇ O₇ P (536.7), FAB-MS (matrix: DMSO/glycerol: m/z=625(M-3H+4Na)⁺, 603 (M-2H+3Na)⁺, 581 (M-H+2Na)⁺, 558 (M-H+Na)⁺, 514, 500,498, 432, 404, 362, 340, 298, 288, 266, 186, 164, 142, 115, 93.

3b

3a was treated in aqueous solution with the ion exchanger (Dowex 50, H⁺form) in order to liberate all acidic groups. The resin was filtered offand then the solution was freeze-dried. 8.5 mg (15.9 μmol) of the sampletreated in this way were dissolved in methanol (2 ml). An excess ofethereal diazomethane solution was added at 0° C. The mixture was leftto stand at 0° C. for 2 h and at 20° C. for 12 h. Evaporation and columnchromatography (5 g of SiO₂, petroleum ether/ethyl acetate 1:1) yielded3b (4.0 mg).

¹ H NMR (80 20 MHz, CDCl₃): δ=3.75 (s, OCH₃ and 2 d, 3J_(H).P =10 and 12Hz, P(OCH₃)₂), 3.20-4.40 (OCH₂ and OCH multiplets), -C₁₃ H₆₃ O₇ P(578.8), MS: m/z (%)=563 (0.1, M-CH₃), 519 (1, M-COOCH₃), 452 (1, M-(CH₃O)₂ P(O)OH), 381 (3, M-C₁₄ H₂₉, breakage of the link between C-8 and C-9of the 25 perhydromoenocinol moiety), 229 (8, a), 212 (6, b), 127 (20),57 (100). ##STR5##

EXAMPLE 7 Antibiotic Activities of the Cleavage Products

An agar dilution test with Mueller-Hinton agar was carried out todetermine the antibacterial activities (Antibiotics in LaboratoryMedicine, V. Lorian, Ed., Baltimore 1986, pages 1-10).

    ______________________________________                                                    Minimum inhibitory                                                            concentration (μg/ml)                                                        Str.        Staph.                                                            pyogenes    aureus  E. coli                                     Cleavage products                                                                           77          503     DC2                                         ______________________________________                                        MA            >100        >100    >100                                        MB            3.125        25     >100                                        MC            >100        >100    >100                                        ______________________________________                                    

EXAMPLE 8 Transglycosylase Assay

The inhibition of the polymerization of the peptido-glycan-sugar chainsby the cleavage products was carried out by the assay described by Izaki(J. Biol. Chem. 243, 3180-3192, 1968) using lipid intermediates from thecell membrane of E. coli K 12.

It emerges from this that moenomycin A (20 μg/ml) inhibits thetransglycosylase reaction by 52.7% and the cleavage product MB inhibitsthe enzyme by 32.9%.

Results of another study of the inhibition of the transglycosylasereaction by moenomycin cleavage products in vitro is shown in thefollowing Table. The values in the Table show the inhibition ofpeptidoglycan-sugar chain synthesis in vitro. Decahydro MB is an analogof MB that has no double bonds in the alkyl chain.

    ______________________________________                                        Inhibition of In Vitro                                                        Peptidoglycan Synthesis                                                                     Final          %                                                Compound      Concentration (μg/l)                                                                      Inhibition                                       ______________________________________                                        MB            12.5           94                                               MA            12.5           67                                               Decahydro MB  12.5           28                                               ______________________________________                                    

EXAMPLE 9 Synthesis of a Transglycosylase Inhibitor Using MA as BuildingBlock

The first step in the synthesis of a transglycosylase inhibitor using MAas a building block is the synthesis of MA-methylester. A mixture ofdried MA (6.0 g) and anhydrous MeOH is stirred at room temperature for30 hours in the presence of Dowex® 50 WX2 (H⁺ -form). Dowex® 50 WX2 isan ion exchanger which acts as an acid and catalyzes the esterification.After 30 hours, the catalyst is removed by filtration, the solvent isevaporated in vacuo and the remaining residue is further purified bycolumn chromatography [silica gel; elution with hexane:ethylacetate(7:1)] in order to obtain MA-methylester. The yield is 4.1 g (=68%).

The second step is the synthesis of the transglycosylase inhibitor2-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glycopyranosyl)-4-O-acetyl-3-O-carbamoyl-1-O{[(R)-2-methyloxycarbonyl-2-(3,8,8,11,14,18-hexamethylnonadec-2,6,11,13,17-pentenyloxy)-ethoxyl]hydroxyphosphoryl}-α-D-galactopyranuronamide,triethylammonium salt, using the MA-methylester. To a solution of1H-1,2,4-triazole (41.3 mg, 0.6 mmol) in 4:1 THF-pyridine (2 ml) isadded 1,1,1-trichloro-2-methyl-prop-2-yl dichlorophosphite (29.6 μl,0.15 mmol) and the mixture is stirred at 0° C. for 20 min with theformation of a colorless precipitate. A solution of2,O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glycopyranosyl)-4-O-(2,2,2-trichlorethoxy)carbonyl-3-O-carbamoyl-α-D-galactopyranuronamide(Moller et al., Tetrahedron, 49:1635-1648 (1993)--compound 6h), (90 mg,0.148 mmol) in 4:1 THF-pyridine (2 ml) is added and stirring at 0° C. iscontinued for 1.5 h. TLC control (hexane-CHCl₃ -methanol 1:1:0.75)indicates a quantitative reaction. A solution of MA-methylester (70 mg,0.15 mmol) in 4:1 THF-pyridine (1 ml) is added and the reaction mixtureis stirred at 0° C. for a further 6 h. Then bis(trimethylsilyl)peroxide(32 μl, 0.15 mmol) is added and the mixture is stirred at 0° C. for 15h. The solvent is evaporated in a stream of argon until only 0.5 ml ofsolution remains. This solution is shown to be free of peroxides.Pyridine (5 ml) is added and the stirred solution is treated with Zn-Cucouple (130 mg) and 2,4-pentanedione (100 μl) for 6 h at 20° C.Filtration, solvent evaporation and MPLC (CHCl₃ -methanol 2.5:1) areused for further purification (78.1 mg, 47%).

Protecting groups (e.g., acetyl or methyl groups) are removed by methodswell known to a person skilled in the art. The acetyl groups can, forexample, be removed as follows:

A solution of the transglycosylase inhibitor (5 mg, 0.0046 mmol) in 2:1methanol-water (double distilled, 0.5 ml) is flushed with argon and then0.3 mol/l LiOH. (92.0 μl, 0.00276 mmol) is added at 0° C. The mixture isstirred at 0° C. for 30 min. The reaction is stopped by addition ofDowex® 50 WX2 (H⁺ form). Stirring at 20° C. for 30 min, filtration,lyophilization, and liquid chromatography (gradient CHCl₃ -methanol2:1→CHCl₃ -methanol-water 20:10:1.5) yields the unprotectedtransglycosylase inhibitor (2.0 mg, 45%).

The methyl group can be removed by the following procedure:

A solution of the transglycosylase inhibitor (20.2 mg, 0.014 mmol) in2:1 methanol-water (double distilled, 2 ml) is flushed with argon, andthen 0.3 mol/l LiOH (371.7 μl, 0.112 mmol) is added at 0° C. The mixtureis stirred at 20° C. for 13 h. The reaction is then stopped by additionof Dowex® 50 WX2 (H⁺ form). Stirring at 20° C. for 30 min, filtration,and lyophilization yields a sample of the unprotected transglycosylaseinhibitor.

The unprotected inhibitor is then used for the assay. Transglycosylaseactivity is assayed by the method of Example 8.

We claim:
 1. A biologically pure culture of Bacillus sp. DSM 4675 andmutants thereof, wherein said mutants are capable of cleavingphosphoglycolipid antibiotics at a phosphoglycosidic linkage.
 2. Abiologically pure culture of Bacillus sp. DSM 4675.